The gene targeting projects that GEF members have conducted in this year include the following scientific areas 1) Human disease modeling: It is often desirable to have mutations of human genetic conditions replicated in mouse so that the diseases can be modeled. In the last year, several such models have been made or are being developed in the NEI Genetic Engineering Core, in collaboration with NEI researchers. Point mutations of the human RPE65 gene are associated with the most severe retinal dystrophy condition, Leber congenital amaurosis (LCA). Three point mutations, which are frequently found in LCA patients, have been separately introduced into the mouse ES cell genome, and all have been microinjected, two of them resulting in germline transmission from chimeric mice. One of the knockin mutations has displayed interesting phenotypes of a delayed onset of retinal degeneration mimicking clinical manifestation of patients with the same mutation. This model system will be a much closer approximation of RPE65-based LCA in human than the total knockout mouse developed over 10 years ago. These engineered mutant mice will serve as a model system for studying the disease, and possibly participate in preclinical therapy trails. Other disease models which have been pursued in the core, and are at different stages of development include: Cep290 knockout for retinal degeneration, which has achieved germline transmission;RPGR point mutation knockin for Retinitis Pigmentosa, and Zfp503 knockout for coloboma, both of which have been microinjected. 2) Reverse genetic confirmation of disease candidate genes: The core supports the disease gene discovery programs by making knockouts and knockins of the candidate genes to establish direct linkage between the genes identified in forward genetic screens and the genetic conditions against which the screens were conducted. Projects in this area include: Cyp4v3 knockout for Bietti crystalline corneoretinal dystrophy, and KLHL7 for Retinitis Pigmentosa, which has been assigned as disease candidate gene by linkage analyses by NEI researchers. 3) Functional genomic studies of genes with interesting expression patterns and predicted to be functionally important in physiology and pathology: Most of the current gene targeting projects are aimed at simply understanding of the functions of various genes relevant to NEI as well as other participating IC research programs. Examples of such include: growth/cytokine genes, which may be critically involved in lens morphogenesis such as KLPH knockout which has displayed phenotypes of cataract development, retinal vascular biology such as Cry-A3/A1 which has been microinjected, PDGF-C and PDGF-D conditional knockouts which have generated an array of interesting phenotypes in neural protection, ocular angiogenesis, and CNV development. Functions of miRNA have also been explored by making loss of function mutations in mice. There are currently 3 miRNA knockout projects at different stages of development in the core. The transgenic mouse projects in which the GEF has been involved this year can be divided into several major categories. Expression of normal or mutant proteins ectopically to help determine their roles in ocular physiology and structure. In this category, 5 constructs were microinjected to investigate retinal degenerations and diseases. Tissue-specific expression of cre recombinase for use in generating conditional gene knockout animal models. This category represented 2 constructs this year;one astrocyte-specific and one retiana-specific. Complex transgenes which include multiple functionalities. This category represented 3 constructs this year During the past year, we have: * worked on 31 different gene targeting projects at various stages * made 16 different constructs for gene targeting in ES cells * made 30 endotoxin-free, large scale DNA preparations of the targeting constructs, and subsequently conducted electroporation experiments with each of the DNA preparations * picked, expanded and crypyopreserved 6,000 ES colonies/clones * expanded 155 positive ES clones, 46 of which were thawed and grown in culture for karyotyping and microinjection * assisted in PCR screening of targeted clones by performing long range PCR (2,400 reactions) * assisted researchers in Southern blot confirmation of homologous recombination * injected 9 ES cell lines into mouse embryos to generate chimeric mice * injected 10 DNA constructs into fertilized mouse oocytes to generate transgenic mice * isolated DNA from 12,303 mouse tail biopsy samples * performed 17,056 PCR reactions to genotype mice in the facility * set up 2,486 matings to propagate mouse lines * overseen weaning, tagging and tail biopsy of 13,104 mice born in the facility * rederived 11 mouse lines * worked on cryopreservation of 63 mouse lines and 18 rat lines, freezing 2 cell stage embryos and sperm * reconstituted for researchers 5 mouse lines from frozen germplasm stock * performed assisted reproduction to save 9 mouse lines from extinction. These services and collaborative services were performed for 16 PIs from 6 NEI labs (LI, LMDB, LRCMB, N-NRL, OGVFB, OSD), plus 6 PIs from 3 other institutes at NIH (NIDCD, NICHD, NINDS). Additional publications to which the NEI Genetic Engineering Core Facility contributed: Xuri Li, Chunsik Lee, Zhongshu Tang, Fan Zhang, Pachiappan Arjunan, Yang Li, Xu Hou, Anil Kumar, Lijin Dong, Cell adhesion &migration. 11/2009;3(4).